Glycan arrays on novel aluminum coated glass slides, including poly-fluorophosphonated aluminum coated glass slides, allow characterization by mass spectrometry without matrix, fluorescence assessment of sugar-protein binding, and identification and study of enzymes with different efficiency and specificity.
Based on the SWISS-PROT protein database, more than 50% of human proteins are predicted to be glycosylated. Carbohydrates often exist on cell surfaces as glycoprotein or glycolipid conjugates and play important structural and functional roles in numerous biological recognition processes, for example, protein folding, secretion and stabilization, viral and bacterial infection, cancer metastasis, inflammatory response, innate and adaptive immunity, and many other receptor-mediated signaling processes. Moreover, there exist many examples in which glycosylation is required for biological activities. Furthermore, many organisms such as sessile plants have evolved specific glycosylation mechanisms to detoxify harmful exogenous xenobiotics.
Despite the increasing awareness of the biological significance of carbohydrates, the study of carbohydrate-protein interactions still encounters much difficulty, largely because of the structure complexity and synthetic difficulty of carbohydrates and the low affinity of their interactions with glycan-binding proteins (GBPs). Typically the monomeric dissociation constant (KD) in a carbohydrate-protein interaction is in the millimolar range; thus, carbohydrate-mediated biological responses are often through multivalent interaction on the cell surface in order to achieve high affinity and specificity.
A major challenge in cell biology is to define the interaction of oligosaccharides and proteins involved in many biological processes. However, pure oligosaccharides are difficult to obtain and there is a need for development of highly sensitive and high-throughput methods for identification and binding study of carbohydrates recognized by various receptors.
Carbohydrate microarrays are a powerful tool for the study of glycobiology and the high-throughput bioassay of epidemic diseases. A fundamental problem of this technology is how to characterize and quantify the oligosaccharides that are covalently bound to the surface. Effective immobilization of sugars on the surface is essential for surviving consecutive substrate washing when evaluating sugar-protein binding. Mass spectrometry (MS) has been reported to be a useful analytical method for the high-throughput characterization of immobilized sugars on porous glass slides.
Although a variety of substrates are commercially available for glycan arrays, they are not suitable for direct mass spectrometric analysis. These substrates include glass and polyethylene terephthalate (PET) coated with amine, carboxylate, N-hydroxysuccinimide (NHS), avidin, epoxy, aldehyde, chelating nickel groups, and so on. In fact, NHS-functionalized glass slides are commonly used for the preparation of glycan arrays. A typical example is that of sugar antigens immobilized on the surface of the glass slide, after which a sugar-binding monoclonal antibody and a fluorescence-tagged secondary antibody were incubated for studies of protein-carbohydrate interaction. Although effective, these glass slides are not ideal for use to characterize the bound sugars by mass spectrometry.
Substrates selected for matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS should be conductive or semiconductive so that a uniform electric field can be produced under high vacuum. Standard stainless-steel plates are usually the choice for loading the analytes.
In MALDI MS, the energy of the pulse laser beam is absorbed by the matrix (miscible organic chemicals) to prevent sample fragmentation. MALDI-TOF MS is an excellent tool for analyzing high-molecular-weight biomolecules. However, the chemicals in the organic matrix interfere with low-molecular-weight oligosaccharides (typically less than 2000 Da); thus, porous silicon was chosen as the substrate for analyzing biomolecules by MS without the addition of matrix chemicals. In desorption-ionization on silicon (DIOS) MS, biomolecules of relatively low molecular weight were identified on the basis of the m/z ratio of the pseudoparent peak from MS.